1. Mechanism of the T286A-Mutant aCaMKII Interactions with Ca2/Ca1modu1in and ATP.
- Author
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Tzortzopoulos, Athanasios and Török, Katalin
- Subjects
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CALMODULIN , *COORDINATES , *CHEMICAL reactions , *PROTEIN kinases , *SMOOTH muscle , *ENZYMES - Abstract
The role of adenosine 5'-triphosphate (ATP) in the activation mechanism of α-Ca2+/calmodulin- dependent protein kinase II (αCaMKII) was investigated using the T286A non-autophosphorylatable mutant of αCaMKII. Characterization of the T286A-αCaMKII mutant revealed kcat = 0.06 ± 0.02 s-1 for the T286A mutant, a 6 (± 2)-fold lower value compared to wild-type αCaMKII with 100 µM smooth muscle myosin light chain (MLC) as substrate. MLC phosphorylation by the T286A mutant and wild-type αCaMKII was cooperative, with Hill coefficients 2.3 ± 0.1 and 2.4 ± 0.3, respectively. Km values for MLC were 96 ± 28 µM with T286A-αCaMKII and 49 ± 29 µM for wild-type αCaMKII. Thus, while the activity of αCaMKII was sensitive to mutation of the Thr286 residue to Ala, the mechanisms of the wild-type and T286A mutant enzyme appeared similar. Kd for Ca2+/calmodulin was 2-fold reduced to 40 nM compared to that of wild-type αCaMKII (75 nM). ATP induced a 9-fold stabilization of Ca2+/calmodulin binding to the T286A mutant enzyme. Fluorescence stopped-flow kinetic experiments revealed that two Ca2+/ calmodulin-enzyme complexes were formed, the first, unaffected by ATP, with association and dissociation rate constants of 2 × 107 M-1 s-1 and 5 s-1, respectively, containing calmodulin in extended conformation. The second complex, in which calmodulin adopted a compact conformation, was formed with association rate constant 3 × 106 M-1 s-1 and dissociation at 0.15 s-1 in the absence and 0.015 s-1 in the presence of ATP. These data show that ATP is involved in the activation mechanism by forming two classes of Ca2+/calmodulin·αCaMKII-ATP complex. It is likely that only one of the complexes is on the activation pathway. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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